Mechanism Study Underlying Anti-angiogenesis Effects Of Two Fucoidans, FP08s2 And NDH01

Sulfated fucoidans, exhibit a wide range of biological activities, such as anti-hyperlipidemia, anti-oxidation, anti-inflammation, immunoregulation and anti-tumor effects. FP08S2 and NDH01 are fucoidans extracted and isolated from two kinds of brown seaweeds, Sargassum fusiforme(Harv.) Setch. and Nemacystus decipiens(Sur.) Kuck, respectively. Bioactivity screening indicated that both FPP08S2 and NDH01 possessed anti-angiogenetic activity.A fucoidfan FP08S2 was isolated and purified from Sargassum fusiforme. Average molecular weight of FP08S2 was 47.5 kD. The backbone of FP08S2 consisted of alternate 1,2-linked α-D-Manp and 1,4-linked β-D-Glcp A. There was a sulfate group on the C-6 of α-1,2-linked manose. The branches contained fucose, xylose, galactose, gulcuronic acid and sulfate. FP08S2 contained fucose majorly and 20.8% sulfate. Bioactivity test suggested that FP08S2 showed excellent anti-angiogenetic activity. Firstly, FP08S2 could significantly inhibit angiogenesis in in vivo chicken choriaollantoic membrane(CAM) model and in vitro HMEC-1(human microvascular endotherlial cell) tube formation model. Secondly, FP08S2 could significantly imped tube formation, migration and invasion, However, it had no impact on cell growth of vascular endothelial cells. Further study indicated that F-actin of HMEC-1 cells was depolymerized after FP08S2 treatment, which would destroy cytoskeleton and inhibit migration, invasion as well as tube formation of HMEC-1 cells.The surface plasmon resonance(SPR) analysis showed that FP08S2 interactedwith both VEGF and VEGFR2. These interactions blocked VEGF and VEGFR2 interaction competitively, suggesting that FP08S2 might target VEGF and its receptorVEGFR2. Importantly, we next confirmed that FP08S2 could inhibit VEGF inducedangiogenesis in vivo by Matrigel plug assay and in vitro by HMEC-1 cells tubeformation assay. In order to understand the roles of VEGF and VEGFR2 in FP08S2 mediated anti-angiogenesis, we then discussed the detailed mechanism and signaling pathway as follows: FP08S2 could bind to VEGF and VEGFR2 so that it could interfere with the interaction between VEGF and VEGFR2. FP08S2 further attenuated VEGFR2 activation to suppress Erk phosphorylation and downregulated the expression of VEGF and its transcription factors(HIF-1α、AP-1 and AP-2).In vivo test showed that FP08S2(0.5 mg/kg and 10 mg/kg) might inhibit xenografted lung caner A549 cells growth after the administration via tail vein injection. However, it had no effect on mice body weights. The immunohistochemistry analysis indicated that FP08S2 inhibited the expression of Ki67 and CD31 in xenografts. In addition, FP08S2 had no significant effect on cell growth of A549 lung cancer cells in vitro. Taken together, these results suggested that FP08S2 might inhibit tumor growth via disrupting tumor angiogenesis in vivo.Another fucoidfan NDH01 was isolated and purified from Nemacystus decipiens. It was a highly branched and acetylized fucoidan, whose average molecular weight was 216 kD. The backbone of NDH01 was fucose-free core, composed of α-D-1,2-Manp and β-D-1,4-Glcp A disaccharide repeat unit. The branches were attached at the C3, C4 and C6 of α-D-1,2-Manp. The sidechain was composed of α-L-1,3,4-Fucp, α-L-1,4-Fucp, α-L-1,3-Fucp and α-L-1,4-Glcp A. The sulfate group was linked to C4 of α-L-1,3,4-Fucp, whereas, acetyl group was branched on C2 of α-L-1,2,3-Fucp. In this study, we showed that NDH01 inhibited cell proliferation, cell migration and tube formation of HMEC-1 cells. Results from SPR assay revealed that NDH01 could strongly interact with BMP4. In addition, it was observed that downstream signaling pathways such as Smad1/5/8, Erk MAPK and FAK pathways were blocked by NDH01 significantly. Further, BMP4 was downregulated by NDH01, which inactivated BMP receptors and the downstream signaling pathways.In summary, we uncovered that two fucoidans FP08S2 and NDH01 could potently inhibit angiogenesis. FP08S2 targeted VEGF/VEGFR2 signaling pathway, while NDH01 targeted BMP4/Smad signaling pathway. According to targeting research, mechanism study and in vivo tumor xenograft model, FP08S2 was demonstrated to inhibit angiogenesis as well as tumor growth both in vitro and in vivo. For NDH01, we provided the first demonstration concerning fucoidan targeting BMP4 directly. NDH01 not only interacted with BMP4 but also downregulated BMP4 expression in order to inhibit Smad signaling pathway and exert angiogenesis effect. Different structure motifs of fucoidans inhibited angiogenesis via targeting different functional proteins. These studies provided novel insights to better understand the actions of fucoidans in angiogenesis and even pave the way for fucoidan-based new drug development.